Transgenic maize plant exhibiting increased yield and drought tolerance

ABSTRACT

The present invention is directed to a transgenic maize plant or a part thereof comprising as transgene a nucleic acid capable of expressing a cell wall invertase or a functional part thereof, preferably a Chenopodium rubrum cell wall invertase or a functional part thereof, wherein as a result of the expression of the cell wall invertase or a functional part thereof the transgenic maize plant exhibits an enhanced tolerance to abiotic stress and/or an increased yield, to a method of producing such transgenic maize plant, to method of enhancing the tolerance to abiotic stress of a maize plant and/or of increasing yield potential of a maize plant, to a nucleic acid capable of expressing a cell wall invertase or a functional part thereof, preferably a Chenopodium rubrum cell wall invertase or a functional part thereof, to a vector comprising such nucleic acid, the use of the nucleic acid or vector for enhancing the tolerance to abiotic stress of a maize plant, for increasing yield potential of a maize plant and/or for protecting a maize plant against abiotic stress, and to a method for production of ethanol or methane from transgenic maize plant or a part thereof of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of International PatentApplication No. PCT/EP2018/052315, filed Jan. 30, 2018, which claimspriority to European Patent Application No. 17153839.0, filed Jan. 30,2017, both of which applications are herein incorporated by reference intheir entireties.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jul. 26, 2019, is named245761_000082_seqlist.txt, and is 15,002 bytes in size.

Plants are exposed during their life term to a series of abiotic stressconditions such as heat stress, frost stress, chilling stress, salinitystress, drought stress etc. Such stress conditions are importantlimiting factors for plant growth and productivity. Thus, exposure ofplants for example to heat and/or drought conditions may typically leadto reduction of yields of plant material such as leaves, seeds, fruitsand other edible or usable products. Such yield reductions representwith economically important plants such as maize, rice or wheat animportant economical factor, whereby especially in many underdevelopedcountries such yield reductions may result in food shortages whichendanger the food supply of the population.

Maize is the most widely produced crop in the world. This cereal isgrown in at least 164 countries around the world with a total productionof more than 1 billion metric tons. Maize is grown at latitudes varyingfrom the equator to slightly above 50 degrees north and south, from sealevel to over 3000 meters elevation, in cool and hot climates, and withgrowing cycles ranging from 3 to 13 months. It is therefore ofimportance for the food supply of the world population that the supplywith maize plants remains at high level. However, especially regionswith extreme weather conditions such as extreme heat, extreme cold,extreme wetness, extreme drought etc. run danger that the food supply isnot ensured, which, in view of obvious weather changes in the lastyears, has become an even more critical subject. Moreover, theimportance of maize as a renewable resource has increased in the lastyears in view of the fact that the combustion of resources such as oil,coal, and natural gas contributes to the warming of the world climateand resources are needed which, due their regrowth, do not contribute toa negative CO₂ balance.

In view of this, it is a scientific demand to provide maize plants andother crop plants which brave the climate in all its forms and otherabiotic factors and, despite heat, chilling, drought, salinity, wetnessetc. consistently provide high yields.

Cell wall invertases, also called extracellular invertases, are crucialenzymes for an appropriate metabolism, growth and differentiation ofplants. They work by hydrolysis of sucrose into glucose and fructoseoutside cells which are subsequently imported into target cells bymonosaccharide transporters. The monosaccharides do not only serve as asource of carbon and energy for plants, but they are also key signalingmolecules that potentially regulate cell division, growth,differentiation, metabolism and resource allocation in plants. Cell wallinvertases are regarded as crucial to supply sink tissues withcarbohydrates via an apoplastic pathway.

Cell wall invertases are known in the art as potentially increasing thegrain yield and biomass of certain plants. Thus, Li et al. (Li B. et.al., Plant Biotechnology Journal, 2013, 11, 1080-1091) disclose theconstitutive overexpression of three cell wall invertase genes (AtCWIN1,OsGIF1 and ZmMn1) in transgenic maize plants leading to an increase ingrain yield. Schweinichen and Büttner (Schweinichen C. and Büttner M.,Plant Biol. (Stuttg), 2005, 7, 469-475) disclose the root-specificexpression of Chenopodium rubrum cell wall invertase in Arabidopsisleading to early flowering and increased biomass of the whole plant,probably due to an extensive root growth. Albacete A. et al., Journal ofExperimental Botany, 2015, 66, 863-878 disclose that fruit-specificexpression of Chenopodium rubrum cell wall invertase in transgenictomato can lead to improved drought tolerance, however they did notobserved an increased shoot weight or leaf area, i.e. biomass.

Despite these successes of increasing plant yields, there is still aneed to provide economically important plants which produce high biomassyield, even under adverse abiotic conditions.

To address this issue, the present inventors succeeded in developingmaize plants which overcome disadvantages of previous maize plants inthat these maize plants show both, an enhanced drought tolerance andbiomass production. Thereby, the present inventors introducedChenopodium rubrum cell wall invertase (CrCIN) into maize plants andfound that these maize plants produced increased yield and had increasedtolerance to drought.

The invention is described in the following, with reference to theclaims.

In the following, the present invention is described in detail. Thefeatures of the present invention are described in individualparagraphs. This, however, does not mean that a feature described in aparagraph stands isolated from a feature or features described in otherparagraphs. Rather, a feature described in a paragraph can be combinedwith a feature or features described in other paragraphs.

The term “comprise/es/ing”, as used herein, is meant to “include orencompass” the disclosed features and further features which are notspecifically mentioned. The term “comprise/es/ing” is also meant in thesense of “consist/s/ing of” the indicated features, thus not includingfurther features except the indicated features. Thus, the product andmethod of the present invention may be characterized by additionalfeatures in addition to the features as indicated.

In a first aspect, the present invention relates to a transgenic maizeplant comprising as transgene i) a nucleic acid capable of expressing aChenopodium rubrum cell wall invertase (CrCIN) or a functional partthereof, ii) the nucleic acid capable of expressing the Chenopodiumrubrum cell wall invertase or the functional part thereof of item i)which is modified by the degeneration of the genetic code, iii) anucleic acid capable of expressing a cell wall invertase or a functionalpart thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%amino acid identity or at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% amino acid homology to the Chenopodium rubrum cell wallinvertase or the functional part thereof of item i), or iv) a nucleicacid capable of hybridizing under stringent conditions with acomplementary sequence of the nucleic acid of any one of items i) toiii), whereby the nucleic acid of item iv) is capable of expressing acell wall invertase, wherein as a result of the expression of theChenopodium rubrum cell wall invertase or a functional part thereof or ahomolog thereof the transgenic maize plant exhibits an enhancedtolerance to abiotic stress and/or an increased yield, optionally ascompared to a reference.

In an embodiment thereof, the nucleic acid is derived from the nucleicacid of any one of items i) to iv) by codon optimization.

In an embodiment of the above, the nucleic acid of item i) comprises thenucleic acid sequence of SEQ ID NO: 3 or encodes the amino acid sequenceof SEQ ID NO: 4.

In an embodiment of the above, the transgenic maize plant comprises astransgene an expression cassette comprising the nucleic acid.

In an embodiment of the above, the nucleic acid or the expressioncassette is stably integrated into the genome of the maize plant or istransiently expressed in the maize plant, for example is present in themaize plant on a vector.

In an embodiment of the above, the expression of the nucleic acid iscontrolled by a promoter, preferably a constitutive promoter.

In an embodiment of the above, the abiotic stress is selected fromdrought, salinity, heat or chilling and/or the yield is biomass yield orgrain yield.

The present inventors have surprisingly demonstrated that Chenopodiumrubrum cell wall invertase is effective in enhancing in a maize planttolerance to drought stress and/or of increasing yield of a maize plant.This is surprising insofar, as the present inventors also demonstratedthat the same gene introduced into wheat plants were not effective inincreasing biomass or grain yield. This shows that the effect of cellwall invertases in general and specifically of Chenopodium rubrum cellwall invertase in a heterologous setting is not predictable.

Thus, by introducing the gene coding for Chenopodium rubrum cell wallinvertase the present inventors were able to enhance in a maize planttolerance to abiotic stress and/or to increase (biomass) yield of amaize plant under normal and/or stress conditions. Specifically, thepresent inventors showed that the gene coding for Chenopodium rubrumcell wall invertase introduced into a maize plant was expressed andexpression of the gene resulted in an enhanced production of leaves, inthe production of higher plants and in the production of maize plantswith a higher drought resistance as compared to a reference.

The transgenic maize plant of the present invention expressesChenopodium rubrum cell wall invertase (CrCIN). The gene encoding theCIN1 cell wall invertase derived from Chenopodium rubrum is known in theart and is, e.g. characterized by the accession number as available fromthe NCBI database (National Centre for Biotechnology Information;National Library of Medicine 38A, Bethesda, Md. 20894, USA;www.ncbi.nih.gov) under the accession number X81792.1 (SEQ ID NO: 1)encoding the protein with the accession number CAA57389.1 (SEQ ID NO:2). Chenopodium rubrum cell wall invertase and the gene encoding thecell wall invertase are not restricted to SEQ ID NOs: 1 and 2, butinclude any Chenopodium rubrum cell wall invertase naturally expressedby Chenopodium rubrum and the gene encoding the Chenopodium rubrum cellwall invertase. Moreover, the transgenic maize plant of the presentinvention comprises a nucleic acid that expresses a “homolog” of aChenopodium rubrum cell wall invertase. A “homolog”, as defined herein,is a cell wall invertase which has an amino acid identity to aChenopodium rubrum cell wall invertase, as exemplarily identified by SEQID NO: 2, of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, orwhich has an amino acid homology to a Chenopodium rubrum cell wallinvertase, as exemplarily identified by SEQ ID NO: 2, of at least 70%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. Thereby, “amino acidhomology” refers to identical or homologous amino acids. Homologousamino acid residues have similar chemical-physical properties, forexample, amino acids belonging to a same group: aromatic (Phe, Trp,Tyr), acid (GIu, Asp), polar (GIn, Asn), basic (Lys, Arg, His),aliphatic (Ala, Leu, lie, VaI), with a hydroxyl group (Ser, Thr), orwith a short lateral chain (GIy, Ala, Ser, Thr, Met). It is expectedthat substitutions between such homologous amino acids do not change aprotein phenotype (conservative substitutions). Alternatively, a“homolog” is a cell wall invertase which is encoded by a nucleic acidwhich is capable of hybridizing under stringent conditions with thecomplementary sequence of the nucleic acid coding for a Chenopodiumrubrum cell wall invertase, such as identified by SEQ ID NO: 2, or withthe complementary sequence of the nucleic acid coding for a cell wallinvertase which has amino acid identity or amino acid homology toChenopodium rubrum cell wall invertase, as identified above.

The Chenopodium rubrum cell wall invertase, such as identified by SEQ IDNO: 2, or a homolog thereof confers on the maize plant an enhancedtolerance to abiotic stress and/or the maize plant harboring a nucleicacid coding for Chenopodium rubrum cell wall invertase, such asidentified by SEQ ID NO: 2, or a homolog thereof has an increased yield,optionally as compared to a reference. Preferably, Chenopodium rubrumcell wall invertase, such as identified by SEQ ID NO: 2, or a homologthereof may not be capable of conferring on a wheat plant into which ithas been transformed tolerance to abiotic stress and/or of increasingthe yield of a wheat plant, more specifically Chenopodium rubrum cellwall invertase or a homolog thereof may not be capable of increasingwheat plant height or grain yield.

As used herein, a “functional part” of a Chenopodium rubrum cell wallinvertase or of a homolog thereof refers to any part of the proteinwhich has the same activity as full-length Chenopodium rubrum cell wallinvertase such as identified SEQ ID NO: 2, namely the functional parthydrolyses sucrose into glucose and fructose. Moreover, the functionalpart confers on the maize plant an enhanced tolerance to abiotic stressand/or the maize plant harboring the functional part has an increasedyield, optionally as compared to a reference. Preferably, the functionalpart may not be capable of conferring on a wheat plant into which it hasbeen transformed tolerance to abiotic stress and/or of increasing theyield of a wheat plant, more specifically the functional part may not becapable of increasing wheat plant height or grain yield.

As used herein, the term “maize plant” means any plant of the speciesZea mays.

As used herein, the term “nucleic acid” may be a DNA, a RNA or a hybridof DNA and RNA. Preferably, the DNA is double-stranded. It may be agenomic DNA comprising intron sequences and possibly regulatorysequences in the 5′ and/or 3′ region or a cDNA without intron sequences.The term “nucleic acid”, as used herein, comprises nucleic acids whichencode Chenopodium rubrum cell wall invertases or a functional partthereof or a homolog thereof, as defined above. Moreover, the term“nucleic acid” comprises a nucleic acid which is modified by thedegeneration of the genetic code of a nucleic acid encoding a naturallyoccurring Chenopodium rubrum cell wall invertase.

As used herein, the term “nucleic acid” is also meant to include a partof a nucleic acid encoding Chenopodium rubrum cell wall invertase or ahomolog thereof, whereby the part of a nucleic acid encodes a functionalpart of a Chenopodium rubrum cell wall invertase or a homolog thereof,as defined above.

The term “degeneration of the genetic code” refers to the degeneracy ofcodons which is a term known in the art and means the redundancy of thegenetic code exhibited as the multiplicity of three-base pair codoncombinations that specify a given amino acid. Thus, the codon coding foran amino acid can be specifically changed without that the amino acid ischanged. This results in a variety of nucleic acids coding for the sameChenopodium rubrum cell wall invertase.

The percentage of “sequence identity” or “sequence homology”, as usedherein, refers to the percentage of amino acid residues which areidentical or homologous, respectively, in corresponding positions in twooptimally aligned sequences. It is determined by comparing two optimallyaligned sequences over a comparison window, where the fragment of theamino acid sequence in the comparison window may comprise additions ordeletions (e.g., gaps or overhangs) as compared to a reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The percentage is calculated by determining thenumber of positions at which the identical or homologous amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Optimal alignment ofsequences for comparison may be conducted by the local homologyalgorithm of Smith T. F. and Waterman M. S., Add APL Math, 1981, 2,482-489, by the homology alignment algorithm of Needleman S. B. andWunsch C. D., J. Mol. Biol., 1970, 48, 443-453, by the search forsimilarity method of Pearson W. R. and Lipman D. J., PNAS, 1988, 85,2444-2448, by the algorithm of Karlin S. and Altschul S. F., PNAS, 1990,87, 2264-2268, modified by Karlin S. and Altschul S. F., PNAS, 1993, 90,5873-5877, or by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection. GAP and BESTFIT are preferably employed to determinethe optimal alignment. Typically, the default values of 5.00 for gapweight and 0.30 for gap weight length may be used.

As used herein, the term “hybridize(s)(ing)” refers to the formation ofa hybrid between two nucleic acid molecules via base-pairing ofcomplementary nucleotides. The term “hybridize(s)(ing) under stringentconditions” means hybridization under specific conditions. An example ofsuch conditions includes conditions under which a substantiallycomplementary strand, such as a strand composed of a nucleotide sequencehaving at least 80% complementarity, hybridizes to a given strand, whilea less complementary strand does not hybridize. Alternatively, suchconditions refer to specific hybridizing conditions of sodium saltconcentration, temperature and washing conditions. As an example, highlystringent conditions comprise incubation at 42° C., 50% formamide, 5×SSC(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate,5×Denhardt's solution, 10× dextran sulphate, 20 mg/ml sheared salmonsperm nucleic acid and washing in 0.2×SSC at about 65° C. (SSC standsfor 0.15 M sodium chloride and 0.015 M trisodium citrate buffer).Alternatively, highly stringent conditions may mean hybridization at 68°C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for16 hours and washing twice with 2×SSC and 0.1% SDS at 68° C. Furtheralternatively, highly stringent hybridisation conditions are, forexample: Hybridizing in 4×SSC at 65° C. and then multiple washing in0.1×SSC at 65° C. for a total of approximately 1 hour, or hybridizing at68° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSAfor 16 hours and subsequent washing twice with 2×SSC and 0.1% SDS at 68°C.

The present inventors showed that the nucleic acid encoding aChenopodium rubrum cell wall invertase or a functional part thereof or ahomolog thereof can be expressed in a maize plant. The expressed cellwall invertase confers on the maize plant an enhanced tolerance toabiotic stress and/or the maize plant exhibits an increased yield.“Tolerance to abiotic stress” means that the introduction and expressionof Chenopodium rubrum cell wall invertase or a functional part thereofor a homolog thereof in a maize plant renders the maize less susceptibleto adverse abiotic conditions, whereby typical stress symptoms due tothe adverse abiotic factors do not occur or occur to a lesser degreethan in a reference. Alternatively or additionally, introduction andexpression of Chenopodium rubrum cell wall invertase or a functionalpart thereof or a homolog thereof in a maize plant increases the yieldof the maize plant, optionally as compared to a reference. “Increasedyield”, as used herein, means that the transgenic maize plant exhibitsan increased growth rate under normal conditions which do not producestress to the plant or abiotic stress conditions, optionally as comparedto a reference. An increased growth rate comprises an increased massproduction of the whole plant or a part thereof such as an increasedmass production of the overground part of the plant, e.g. of stem,leaves, florescence, cobs, and/or grains etc., and/or an increased massproduction of the underground part of the plant. The “increased massproduction” may include any part of the transgenic maize plant andrefers in particular to the stem, leaves, cobs and/or grains. “Increasedyield” also comprises a prolonged growth and survival, also resulting inan increased mass production.

As used herein, the term “reference” may refer to a maize plant of thesame genotype as the transgenic maize plant of the present inventionwhereby the reference does not comprise the transgene encodingChenopodium rubrum cell wall invertase or a functional part thereof or ahomolog thereof. Reference experiments including (a) reference maizeplant(s) may be conducted parallel to the experiments for testing theproperties of the transgenic maize plants of the present invention.However, reference experiments may also be conducted at a different timepoint under comparable conditions and the results may be compared afterall experiments are finished. Alternatively, the “reference” may be aspecific (pre)determined measure of yield or of a symptom such as thepercentage of leaves showing leaf rolling symptom under droughtconditions which characterizes a maize plant as having tolerance to anabiotic stress factor or as having no tolerance to an abiotic stressfactor. For example, a reference measure may be an already determinedmeasure or a publicly available measure which provides to the skilledperson a threshold measure and helps him/her to decide that a transgenicmaize plant is tolerant or not tolerant to an abiotic stress factor orhas an increased yield, dependent of whether the measure of thetransgenic maize plant is below or above this measure. Based on thisreference measure (e.g. number of leaves with rolling symptoms), theskilled person can then identify a maize plant as being tolerant to astress factor if the maize plant has a lower number of leaves withrolling symptoms than the reference measure or as having increased yieldif the maize plant indicates a higher yield than the reference measure.Thereby, the maize plant(s) used for establishing the reference measuredoes not need to be, but may be, a maize plant of same genotype as thetransgenic maize plant. For example, the reference maize plant(s) may be(a) maize plant(s) which has(ve) a degree of tolerance to an abioticstress factor which reflects the average tolerance degree of apopulation of maize plants adapted to a specific environment. Theskilled person who wants to develop a maize plant better adapted to thespecific environment may use this measure as reference and develop atransgenic maize plant which exhibits a better measure and which is,therefore, better adapted to the specific environment. Or the referencemaize plant(s) may be (a) maize plant(s) with a certain degree oftolerance to an abiotic stress factor, and it is an object to generate atransgenic maize plant which has a higher degree of tolerance to theabiotic factor. Likewise, the skilled person may want to develop atransgenic maize plant with a high yield under specific conditions, andmay use the comparison with a reference measure to determine whether thetransgenic maize plant meets the object.

For determination whether a transgenic maize plant shows “tolerance toabiotic stress” or “increased yield”, CrCIN transcript and/or proteinexpression and/or expression level from the transgene may be determined,according to methods known in the art. Thus, the determination whether atransgenic maize plant harboring a Chenopodium rubrum cell wallinvertase or a functional part thereof or a homolog thereof hastolerance to abiotic stress or increased yield does not necessarilyrequire the comparison with a reference. The present inventors havefound that introduction and expression of the Chenopodium rubrum cellwall invertase results in an increase in yield and increased toleranceto abiotic stress factors, as is shown in the exemplary part of thepresent specification.

The term “abiotic stress” or “abiotic stress conditions” refers tostress conditions for the maize plant arising from abiotic, i.e.non-living, factors. Such abiotic factors include drought, salinity(concentration of salt), heat or chilling. While not being want to bebound by the following, it may be assumed that the effect of theChenopodium rubrum cell wall invertase or a functional part thereof or ahomolog thereof in the maize plant is related to an increasedcarbohydrate pool, which is generated due to the increased activity ofChenopodium rubrum cell wall invertase or a functional part thereof or ahomolog thereof in the maize plant where the enzyme is overexpressed.Sugars which are generally known to have a protective effect againstosmotic stress may result in a molecular cellular phenotype in the maizeplant which protects the maize plant from stress conditions such asdrought, salinity and/or heat conditions at which osmotic events play arole. Moreover, sugars which are generally known to have protectiveeffect against chilling or frost temperature impacts may result in amolecular cellular phenotype which protects the maize plant from stressconditions such as chilling.

In a preferred embodiment of the present invention, the nucleic acid ascomprised by the maize plant according to the present invention is codonoptimized. Once a cell wall invertase has been selected fortransformation of a maize plant, the codons may be modified and adaptedto the specific requirements of the host in order to maximizeexpression. Codon optimization of a nucleic acid for expression inheterologous host cells is known to those skilled in the art. There arenumerous commercial providers that have developed algorithms thatconsider relevant transcription and translation optimization parametersand deliver a nucleic acid sequence configured to the requirements ofnucleic acid and host. For example, codon optimization can be effectedby the GeneOptimizer™ software, GeneArt, ThermoFisher Scientific. Apreferred nucleic acid, as comprised by the present invention, is thecodon optimized sequence of SEQ ID NO: 3 derived from SEQ ID NO: 1encoding the polypeptide of SEQ ID NO: 4. The codons are especiallyadapted to the codon usage in maize plants.

The term “expression of” or “expressing” means (1) the transcription ofa nucleic acid as comprised by the present invention into an RNA or mRNAand/or (2) the translation of the RNA or mRNA into Chenopodium rubrumcell wall invertase or a functional part thereof or a homolog thereof.

As used herein, an “expression cassette” is a nucleic acid moleculewhich is composed of one or more open reading frames or geneticsequences which are expressed into (a) protein(s) in a maize plant intowhich the expression cassette has been introduced and regulatoryelement(s) in the 5′ and optionally 3′ position controlling theirexpression. Thus, an expression cassette may contain a promoterregulatory sequence, also designated promoter, operably linked to anopen reading frame or another genetic sequence, and a 3′ terminatorregulatory region that may contain a polyadenylation site. The promoterdirects the machinery of the cell to make RNA and/or protein. Theregulatory element(s) may be from the cell wall invertase nucleic acidwhich is introduced into the maize plant or may be from different genes,as long as the regulatory element(s) is(are) able to function in themaize plant. Moreover, the regulatory element(s) in the 5′ position maybe derived from the same gene as the regulatory element(s) in the 3′position or may be derived from different genes. As used herein,“operably linked” means that expression of the linked nucleic acidsequences occurs in the maize plant. An expression cassette may be partof a vector used for cloning and introducing the nucleic acid into acell.

For introducing the nucleic acid molecule capable of expressing aChenopodium rubrum cell wall invertase or a homolog thereof or afunctional part thereof into a cell, the nucleic acid molecule or theexpression cassette harboring the nucleic acid may be inserted into avector. Vectors which harbor a nucleic acid molecule are known to thosein the art. In addition to the nucleic acid molecule, the vector maycomprise regulatory element(s) in the 5′ and optionally in the 3′positions which are able to function in a maize plant. The regulatoryelement(s) are preferably heterologous to the Chenopodium rubrum cellwall invertase or a functional part thereof or a homolog thereof. Thus,the vector may comprise a promoter regulatory sequence operably linkedto the nucleic acid molecule, and optionally a terminator regulatorysequence. Preferably, the vector is a shuttle vector for transformationinto Agrobacterium tumefaciens and subsequent transfer of the nucleicacid molecule encoding a Chenopodium rubrum cell wall invertase or afunctional part thereof or homolog thereof into maize plant cells byinfection of the maize plant by the transformed Agrobacteriumtumefaciens. More preferably, the vector is a binary vector which is astandard tool in the transformation of higher plants mediated byAgrobacterium tumefaciens. It is composed of the borders of T nucleicacid, multiple cloning sites, replication functions for Escherichia coliand Agrobacterium tumefaciens, selectable marker genes, reporter genes,and other accessory elements that can improve the efficiency of and/orgive further capability to the system. Another more preferred vector isa super-binary vector that carries additional virulence genes from a Tiplasmid, and exhibits very high frequency of transformation, which isvaluable for recalcitrant plants such as cereals. A number of usefulvectors are available in the art. Especially preferred is a binaryvector which comprises the ubiquitin promoter of maize (e.g., U.S. Pat.Nos. 5,510,474 A, 6,020,190 A, 6,054,574 A, 6,878,818 B1, 6,977,325 B2)and the nos terminator sequence of Agrobacterium tumefaciens or the 35Sterminator sequence of cauliflower mosaic virus as transcriptionregulatory sequences and preferably extended by a herbicide resistancegene (e.g. pat gene for conferring Basta resistance (e.g., U.S. Pat. No.7,112,665 B1)) and/or the spectinomycin resistance gene as selectablemarker genes.

According to the invention, the term “promoter regulatory sequence” or“promoter” is intended to mean any promoter of a gene that can beexpressed in a maize plant. Such promoter may be a promoter which isnaturally expressed in the maize plant or is of fungal, bacterial, orviral origin. The promoter may include a constitutive promoter, a tissuespecific promoter, or an inducible promoter, whereby constitutiveexpression is preferred. A number of suitable promoters are available inthe art. For example, a constitutive promoter useful in the invention isthe ubiquitin promoter from maize. Another promoter is the Act-1promoter from rice (e.g., U.S. Pat. No. 5,641,876 A). The NCR promoterfrom soybean chlorotic mottle virus (SoyCMV) (Hasegawa, A., et al. “Thecomplete sequence of soybean chlorotic mottle virus DNA and theidentification of a novel promoter.” Nucleic acids research 17.23(1989): 9993-10013.) has also been shown to be useful inmonocotyledonous plants. Further useful promoters are the 35S CaMV(Franck A. et al., 1980, Cell 21:285-294) and the 19S CaMV promoter fromcauliflower mosaic virus (U.S. Pat. No. 5,352,605; WO 84/02913) or plantpromoters like those from the Rubisco small subunit (U.S. Pat. No.4,962,028).

The promoter used in the method of the invention may be an induciblepromoter. An inducible promoter is a promoter that is capable ofdirectly or indirectly activating transcription of a nucleic acidsequence in response to an inducer. In the absence of an inducer, thenucleic acid sequence will not be transcribed. Inducible expression maybe desirable. Stimuli for inducible promoters are of different kind andinclude environmental conditions such as light, temperature and/orabiotic stress conditions such as water stress, salinity stressconditions, cold stress or heat stress. Other types of stimuli forinducible promoters are hormones (for example gibberellin, abscisicacid, jasmonic acid, salicylic acid, ethylene, auxin) or chemicals(tetracycline, dexamethasone, estradiol, copper, ethanol, andbenzothiadiazol). Thus, the expression of Chenopodium rubrum cell wallinvertase or a functional fragment thereof or a homolog thereof underspecific inducive conditions, preferably under abiotic stressconditions, results in the protection of the maize plant by preventingstress symptoms and allowing the formation of mass. Inducible promotersare e.g. promoters which are benzyl sulfonamide inducible (EP 0 388186), tetracyclin inducible), Gatz C. et al., Plant J. 2, 1992:397-404), abscisic acid inducible (EP 0 335 528) or ethanol orcyclohexenol inducible (WO 93/21334).

In addition to a promoter sequence, an expression cassette or vector mayalso contain a terminator downstream of the structural gene to providefor efficient termination. According to the invention, the term“terminator” or “terminator regulatory sequence” is intended to mean anysuch sequence that is functional in terminating expression of a nucleicacid in a maize plant, also optionally comprising polyadenylationsequences. The terminator may be obtained from the same gene as thepromoter sequence or may be obtained from a different gene. Thus, it maybe of viral origin such as the CaMV 35S terminator which is thepreferred one, of bacterial origin such as the octopine synthase or thenopaline synthase terminator of Agrobacterium tumefaciens, or of plantorigin such as a histone terminator. Polyadenylation sequences include,but are not limited to, the Agrobacterium octopine synthase signal.

The term “introducing” or “introduction”, as used herein, meansinserting a nucleic acid into a maize plant by any means known in theart, such as “transformation” using non-viral introduction methods or“transduction” using viral-mediated gene transfer. For introducing thenucleic acid molecule into a maize plant, numerous methods are known inthe art (see, for example, Miki et al., “Procedures for IntroducingForeign nucleic acid into Plants” in Methods in Plant Molecular Biologyand Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press,Inc., Boca Raton, 1993), pages 67-88). In addition, expression vectorsand in vitro culture methods for plant cell or tissue transformation andregeneration of plants are available (see, for example, Gruber et al.,“Vectors for Plant Transformation” in Methods in Plant Molecular Biologyand Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press,Inc., Boca Raton, 1993), pages 89-119).

A preferred method applied in the present invention is transformation ofthe nucleic acid molecule, expression cassette or vector harboring thenucleic acid molecule by the use of bacteria of the Agrobacterium genus,preferably by infection of cells or tissues of a maize plant with A.tumefaciens (Knopf U. C., 1979, Subcell. Biochem. 6: 143-173; Shaw C. H.et al., 1983, Annu. Rev. Genet. 16: 357-384; Tepfer M. and Casse-DelbartF., 1987, Microbiol. Sci. 4 (1): 24-28). For example, the transformationof maize plant cells or tissues with Agrobacterium tumefaciens iscarried out according to the protocol described by Hiei Y. et al. (1994,Plant J. 6 (2): 271-282).

Another method for introducing a nucleic acid into a maize plant is thebiolistic transformation method, wherein cells or tissues are bombardedwith particles onto which the nucleic acid, expression cassette orvector as comprised by the invention are adsorbed (Bruce W. B. et al.,1989, Proc. Natl. Acad. Sci. USA 86 (24): 9692-9696; Klein T. M. et al.,1992, Biotechnology 10 (3): 286-291; U.S. Pat. No. 4,945,050). A furthermethod is the widely used protoplast transformation. Therefor, plantcells are separated by pectinases and subsequently, the cell wall isdegraded to generate protoplasts. For transformation, polyethyleneglycol may be added or electroporation may be applied. Other methods arebringing the plant cells or tissues into contact with polyethyleneglycol (PEG) and the nucleic acid, expression cassette or vector of theinvention (Chang S. and Cohen S. N., 1979, Mol. Gen. Genet. 168 (1):111-115; Mercenier A. and Chassy B. M., 1988, Biochimie 70 (4):503-517). Electroporation is another method, which consists ofsubjecting the cells or tissues to be transformed and the nucleic acid,expression cassette or vector as comprised by the invention to anelectric field (Andreason G. L. and Evans G. A., 1988, Biotechniques 6(7): 650-660; Shigekawa K. and Dower W. J., 1989, Aust. J. Biotechnol. 3(1): 56-62). Another method consists of directly injecting the nucleicacid, expression cassette or vector as comprised by the invention intothe cells or the tissues by microinjection (Gordon and Ruddle, 1985,Gene 33 (2): 121-136). Another method for physical delivery of a nucleicacid to plants is sonication of target cells (Zhang et al.,Bio/Technology 9: 996 (1991)). Alternatively, liposome or spheroplastfusion may be used to introduce the nucleic acid, expression cassette orvector as comprised by the invention into plants (Deshayes et al., EMBOJ., 4: 2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987)). Direct uptake of a nucleic acid into protoplasts usingCaCl₂ precipitation, polyvinyl alcohol or poly-L-ornithine has also beenreported (Hain et al., Mol. Gen. Genet. 199: 161 (1985); Draper et al.,Plant Cell Physiol. 23: 451 (1982)).

The selection step for identifying a transgenic maize plant comprisingthe nucleic acid can be carried out via a selectable gene present on thevector, as referred to above. The selectable gene may comprise anoperably linked promoter regulatory sequence and possibly a terminatorregulatory sequence that are functional in maize cells. Among theselectable markers that can be used in the present invention, referenceis made to genes for resistance against antibiotics, such as thespectinomycin resistance gene, the hygromycin phosphotransferase gene,the neomycin phosphotransferase II gene inducing resistance againstkanamycin, or the aminoglycoside 3′-adenyltransferase gene, the bar gene(White J. et al., Nucl. Acids Res., 1990, 18: 1062) for tolerance tobialaphos, the EPSPS gene (U.S. Pat. No. 5,188,642) for tolerance toglyphosate or the HPPD gene (WO 96/38567) for tolerance to isoxazoles,genes encoding identifiable enzymes, such as the GUS enzyme, GFP proteinor genes encoding pigments or enzymes regulating pigment production inthe transformed cells. Such selectable marker genes are in particulardescribed in patent applications WO 91/02071, WO 95/06128, WO 96/38567,and WO 97/04103. In a preferred embodiment, the spectinomycin resistancegene and the pat gene are used as the selectable genes on a binaryvector in the present invention.

Marker gene free transformation is another alternative to transfer thenucleic acid, expression cassette or vector, as referred to above, intoa maize plant.

In one embodiment, the nucleic acid or expression cassette is stablyintegrated into the genome of the transgenic maize plant, preferablyinto a chromosome of the plant such as the nuclear, plastid and/ormitochondrial chromosome. Integration can, however, also occur into anextrachromosomal element. By stable integration into the genome of aplant, the nucleic acid sequences can be passed to subsequentgenerations of the transgenic plant. Stable integration and passing tonext maize plant generations is preferred in the present invention.Using the Agrobacterium tumefaciens mediated transformation method ofplants as the preferred transformation method, the Chenopodium rubrumcell wall invertase nucleic acid is stably integrated into the maizeplant genome. Alternatively, the nucleic acid or expression cassette orvector harboring the nucleic acid or expression cassette may beconverted into an autonomous replicon. Alternatively, the nucleic acidmolecule or expression cassette is present within the plant cell on thevector used to introduce the nucleic acid molecule and is not stablyintegrated into the genome of the plant, or the nucleic acid istransiently expressed such as transformed mRNA. Therefore, the nucleicacid sequences may not be passed to subsequent generations of the maizeplant.

The term “heterologous”, as used herein, refers to conditions whereinmolecules are present in environments under which they are not naturallypresent. For example, a nucleic acid molecule which is expressed in ahost cell in which it is not naturally expressed is a heterologousnucleic acid. Consequently, the host cell is then a heterologous hostcell. Heterologous regulatory elements are those which are linked tonucleic acid molecules to which they are not naturally linked.

A “transgenic maize plant”, as used herein, refers to a maize plantwhich contains a nucleic acid capable of expressing a Chenopodium rubrumcell wall invertase or a functional part thereof or a homolog thereofintegrated into its nuclear genome or organelle genome or being presenton an autonomous replicon or on the vector used to introduce the nucleicacid as comprised by the present invention or being present as merecoding sequence without other elements. This term encompasses furtherthe offspring generations such as T1, T2 or consecutive generations, aswell as crossbreeds thereof with non-transgenic or other transgenicplants. The transgenic maize plant advantageously contains at least onecopy of the nucleic acid as comprised by the invention.

Expression of Chenopodium rubrum cell wall invertase or a functionalpart or a homolog thereof in a maize plant enhances tolerance to abioticstress conditions. Preferred “abiotic stress” against which thetransgenic maize plant of the present invention exhibits enhancedtolerance includes drought, salinity (concentration of salt), heatand/or chilling.

“Drought” or “drought conditions” mean conditions of water deficiencyarising from a long period of low or no water supply (water stresscondition), especially conditions that adversely affect growing and/orliving conditions of a maize plant. Under drought conditions, the plantwill show symptoms of injury such as wilting, leaf browning and/or leafrolling, growth is hampered and the plant will eventually die. Droughtconditions can be generated by growing a maize plant of the V2, V3, V4,V5, V6, V7, or V8 (according to Leaf Collar Method described below)stage for one week in ¼ strength Hoagland Solution and then treating itfor one day in 25% PEG6000. “Tolerance to drought”, as used herein, maymean that the transgenic maize plant shows significantly reduced leafrolling symptoms under drought conditions such as treatment of plantswith Hoagland Solution and 25% PEG6000. “Significantly reduced” meansthat the percentage of leaves with rolling symptoms is reduced ascompared to a reference by at least 20, 30, 40, 50, 60, 70, 80, 90, 95or 100%. Alternatively, a maize plant is tolerant to drought if at themost 60, 50, 40, 30, 20, 10 or 5 or less % of the leaves of the maizeplant in the V2, V3, V4, V5, V6, V7, V8 and/or VT (fully mature plantwith inflorescence) stage show rolling symptoms if kept under droughtconditions.

“Salinity” or “salinity conditions” mean conditions of highconcentration of salt such as 100 mM NaCl solution for irrigation,especially in the air and/or in the soil, especially conditions thatadversely affect growing and/or living conditions of a maize plant. Theability of plants to tolerate salt is determined by multiple biochemicalpathways that facilitate retention and/or acquisition of water, protectchloroplast functions, and maintain ion homeostasis. Essential pathwaysinclude those that lead to synthesis of osmotically active metabolites,specific proteins, or certain free radical scavenging enzymes thatcontrol ion and water flux and support scavenging of oxygen radicals orchaperones. The cause of cell wall invertases to protect a maize plantfrom adverse salinity effects may lie in their ability to synthesizeosmotically active compounds. Under salinity conditions, the yield ofthe maize plant will be lower than under non-salinity conditions. Underextended and/or very high salinity conditions, the maize plant willeventually die. “Tolerance to salinity” may mean that the transgenicplant of the V2, V3, V4, V5, V6, V7, or V8 stage survives and/or growsunder salinity conditions as compared to a reference which does nolonger grow or grows to a lesser degree, whereby under very highsalinity conditions and/or over an extended period of salinity, themaize plant will eventually die. By “survives” is meant that thetransgenic maize plant survives for a longer period of time, such as atleast 10, 11, 12, 13 or more days, than the reference. By “grows” ismeant that the increase in yield of the whole maize plant or of partsthereof such as stem, leaves, cobs or grains is at least 20, 30, 40, 50,60, 70, 80, 90 or 100%, as compared to the yield of a control.

“Heat” or “heat conditions” mean conditions under high temperature suchas ca. 33-40° C. at ear level along a 15-days pre-anthesis period,especially conditions that adversely affect growing and/or livingconditions of a maize plant. Rate of plant growth and development isdependent upon the temperature surrounding the plant. Extreme heatevents occurring during the vegetation period seems to have the mostdramatic impact on plant productivity; whereby extreme heat may causereduction in grain yield. In general, extreme high temperatures duringthe reproductive stage may affect pollen viability, fertilization, andgrain formation. Chronic exposures to extreme temperatures during thepollination stage of initial grain set will reduce grain yieldpotential. Acute exposure to extreme events may be most detrimentalduring the reproductive stages of development (Hatfield J. L. andPrueger J. H., 2015, Weather and Climate Extremes, 10: 4-10). Bearing inmind that temperature and extreme temperature events are expected toincrease due to the warming of world climate, the development of maizeplants with an enhanced tolerance to heat stress conditions seems to bean urgent need. “Tolerance to heat”, as used herein, may mean that thetransgenic plant of the V2, V3, V4, V5, V6, V7, or V8 and pollinationstage survives and/or grows under heat conditions as compared to areference which does no longer grow or grows to a lesser degree. By“survives” is meant that the transgenic maize plant survives for alonger period of time, such as at least 10, 11, 12, 13 or more days,than the reference. By “grows” is meant that the increase in yield ofthe whole maize plant or of parts thereof such as stem, leaves, cobs orgrains is at least 20, 30, 40, 50, 60, 70, 80, 90 or 100%, as comparedto the yield of a control.

“Chilling” or “chilling conditions” mean conditions under chillingtemperature such as under 10° C. but above the freezing point,especially conditions that adversely affect growing and/or livingconditions of a maize plant. Chilling may cause damage (chlorosis) andinterrupts the pathways for nutrients and water to flow. Under chillingconditions, the plant will produce less yield. “Tolerance to chilling”may mean that the transgenic plant of the V2, V3, V4, V5, V6, V7, V8(????) stage survives and/or grows under chilling conditions as comparedto a reference which does no longer grow or grows to a lesser degree. By“survives” is meant that the transgenic maize plant survives for alonger period of time than the reference. By “grows” is meant that theincrease in yield of the whole maize plant or of parts thereof such asstem, leaves, cobs or grains is at least 20, 30, 40, 50, 60, 70, 80, 90or 100%, as compared to the yield of a control.

It will be understood by those skilled in the art that, due to the largenumber of different maize varieties that are grown under a broadspectrum of climate and other abiotic conditions, it is difficult toindicate specific values with respect to drought, salinity, heat orchilling, such as days of drought, degree of salinity or height oftemperature, which guide the skilled person under which conditionstolerance to an abiotic factor should be tested. For example, maizeplants with high drought tolerance will need stronger drought conditionsthan maize plants with a lower drought tolerance in order to assesswhether the corresponding transgenic maize plant shows higher toleranceor will produce higher yield. Therefore, the test conditions will dependon the maize plant used for inserting the transgene and/or on thepurpose for which the transgenic maize plant will be used.

Due to the fact that introduction and expression of Chenopodium rubrumcell wall invertase results in an increase in yield of the transgenicmaize plant under normal and drought conditions and in a droughttolerant phenotype, it is not necessarily required that a transgenicmaize plant which expresses a Chenopodium rubrum cell wall invertase ora functional part thereof or a homolog thereof is compared to areference, as referred to herein, if it should be determined whether thetransgenic maize plant has tolerance to abiotic stress such as drought,salinity, heat and/or chilling. It may be sufficient to determineexpression of the Chenopodium rubrum cell wall invertase or a functionalpart thereof or a homolog thereof, e.g. by determining the amount oftranscript or protein, in order to detect that tolerance to the abioticstress factors drought, salinity, heat and/or chilling exists.

Resistance to an abiotic stress factor may be determined by exposing thetransgenic maize plant to an abiotic stress factor and determining thedegree of stress factor symptoms and/or yield. The obtained measures maybe compared to a reference. Resistance may also be detected bydetermining expression or expression level of the transcript expressedfrom the transgene as comprised by the present invention.

In a second aspect, the invention relates to a plant cell, a tissue, aharvestable part or a seed of the transgenic maize plant of the presentinvention, wherein the plant cell, the tissue, the part or the seedcomprises the transgene as comprised by the present invention.

In principle, any part, tissue or organ of a maize plant is includedwithin the present invention to comprise as a transgene a nucleic acidencoding a Chenopodium rubrum cell wall invertase or a functional partthereof or a homolog thereof. Thus, shoot vegetative organs/structures,e.g., leaves, stems, roots, flowers or floral organs/structures, e.g.bracts, sepals, petals, stamens, carpels, anthers or ovules; seed,including embryo, endosperm or seed coat; grain or the mature ovary;plant tissue, e.g. vascular tissue or ground tissue; or cells, e.g.guard cells, egg cells or trichomes; or progeny of the same are includedwithin the present invention. The term “cell” refers to a cell or cellaccumulation within the plant as well as to an isolated cell or isolatedcell accumulation. A cell may have a cell wall or may be a protoplast.The present invention also relates to a seed which comprises the nucleicacid, expression cassette or vector as comprised by the presentinvention. Preferably, the seeds of a transgenic maize plant retain thenucleic acid, expression cassette or vector as comprised by theinvention, so that the new plants generated from a seed continues tocomprise the nucleic acid, expression cassette or vector.

A “harvestable part” is any part of the plant which can be harvested andused by man. Preferably, the harvestable part may be the wholeoverground part of the maize plant which can be cut, possibly fermentedand used as animal food in animal breeding or in biogas plants as energysource for generating energy providing substances such as biofuel suchas ethanol or methane. Preferably, the harvestable part may be the cob,especially the grains, which are used for nutrition of man and animal.

In a third aspect, the invention relates to a method of producing atransgenic maize plant, comprising the steps of introducing into atleast a cell of a maize plant the nucleic acid or the expressioncassette or the vector as comprised by the invention, and regeneratingthe transgenic maize plant from the at least one cell.

As used herein, “regenerating” or “regeneration” means a process ofgrowing an entire maize plant from a single cell, a group of cells, apart of the maize plant or a tissue of the maize plant. The skilledperson knows methods of introducing nucleic acid into at least a cell ofthe maize plant and growing a maize plant therefrom. “At least a cell”means a single cell, a group of cells, a part of the maize plant or atissue of the maize plant.

In a fourth aspect, the invention relates to method of enhancing thetolerance to abiotic stress of a maize plant and/or of increasing yieldpotential of a maize plant, comprising the steps of introducing into atleast a cell of a maize plant the nucleic acid or the expressioncassette or the vector as comprised by the invention, and causingexpression of the nucleic acid, the expression cassette, or the vector.

As used herein, the term “causing expression” means that under theconditions, under which the plant is kept and/or cultivated,transcription of the nucleic acid introduced into the maize plantoccurs. For example, if the promoter is a constitutive promoter,expression occurs consistently, whereas in case the promoter is aninducible promoter, the activity of the promoter can be induced by thepresence or absence of specific biotic or abiotic factors.

As used herein, the term “yield potential” means the capability of thetransgenic maize plant to increase yield. By expression of a Chenopodiumrubrum cell wall invertase or a functional part thereof or a homologthereof, the capability is conferred on the maize plant that its yieldcan be increased.

In a fifth aspect, the invention relates to the use of the nucleic acidor the expression cassette or the vector as comprised by the inventionfor enhancing the tolerance to abiotic stress of a maize plant, forincreasing yield potential of a maize plant and/or for protecting amaize plant against abiotic stress.

As used herein, “protecting a maize plant against abiotic stress” meansconferring resistance against abiotic stress on the maize plant. Aresistant maize plant is not damaged by abiotic stress factors or isdamaged to a lesser degree as compared to a reference. Resistance may bedetermined as tolerance to abiotic stress is determined. This includesthat resistance may be determined by determining transcript and/orprotein expression or expression level from the transgene.

In an embodiment, in the method of the fourth aspect or the use of thefifth aspect the abiotic stress is selected from drought, salinity, heator chilling, and/or the yield potential is biomass yield potential orgrain yield potential

The term “biomass yield potential” or “grain yield potential” has themeaning as referred to above with respect to “yield potential”, therebyreferring to biomass yield or grain yield, respectively.

The term “biomass” generally refers to organic matter derived from aplant. The term “biomass” can be used for a source of energy and doesnot refer to food or feed. Thus, as used herein, the term “biomass”refers to the parts of the maize plant, usually the overground partssuch as the whole overground maize plant, which can be used as an energysource by converting it to various forms of biofuel such as ethanol ormethane.

In a sixth aspect, the invention relates to the nucleic acid which isderived from a nucleic acid encoding Chenopodium rubrum cell wallinvertase or a functional part thereof or a homolog thereof as comprisedby the present invention by codon optimization, preferably wherein thenucleic acid comprises the nucleic acid sequence of SEQ ID NO: 3 orencodes the amino acid sequence of SEQ ID NO: 4. The invention alsorelates to an expression cassette comprising said nucleic acid or avector comprising said nucleic acid or expression cassette.

In a seventh aspect, the invention relates to a vector comprising thenucleic acid as defined or the expression cassette as defined in thepresent invention.

In an eighth aspect, the invention relates to a method for production ofethanol or methane comprising the following steps: cutting thetransgenic maize plant or harvestable part according to the presentinvention, optionally treating the cut maize plant or the cutharvestable part with an ensilage agent, optionally storing the cutmaize plant or the cut harvestable part optionally treated with anensilage agent, and producing ethanol or methane from the cut maizeplant or the cut harvestable part by anaerobic digestion.

The eighth aspect serves to provide a method by which the transgenicmaize plant is used as an energy source for providing biofuel such asethanol or methane which are used in petrol, for heating, for obtainingelectricity etc. The processes for obtaining energy from maize plantsare known in the technical field of biogas recovery where cut maize orother plant material is stored and fermented in a process calledensilage with the help of anaerobic bacteria.

Treatment of the cut biomass with an ensilage agent serves to improvethe ensilaging result. By adding powerful lactic acid bacteria or otherbacteria useful for anaerobic digestion of the biomass and/or chemicalagents, undesired bacteria such as butyric acid generating bacteria areinhibited. Chemical agents may be sodium nitrite or hexamine forreducing undesired bacteria such as butyric acid generating bacteria, orsodium benzoate or potassium sorbate for preventing the growth of yeastsand mildews. Thus, failed anaerobic digestions and/or after-warmingprocesses can be prevented and the anaerobic digestion process can becontrolled.

By “storing the cut maize plant or cut harvestable part” is meant theplacing in a container, silo or pit and compressing it so as to leave aslittle oxygen as possible or keeping it under anaerobic conditions inorder to avoid growth of aerobic bacteria. Storing is preferablyperformed under suitable conditions regarding suitable temperature,moisture, low or no oxygen etc. to allow anaerobic digestion. Theskilled person knows the conditions and devices which are to be used forstorage and anaerobic digestion.

The present invention discloses a method of conferring on a maize planttolerance to abiotic stress, comprising the following steps: introducinginto at least a cell of a maize plant a nucleic acid capable ofexpressing a cell wall invertase or a functional part thereof or ahomolog thereof, an expression cassette comprising the nucleic acid or avector comprising the nucleic acid or the expression cassette, andcausing expression of the nucleic acid, the expression cassette, or thevector.

The present invention discloses the use of a nucleic acid capable ofexpressing a cell wall invertase or a functional part thereof or ahomolog thereof, an expression cassette comprising the nucleic acid or avector comprising the nucleic acid or the expression cassette forconferring on a maize plant tolerance to abiotic stress or forprotecting a maize plant against abiotic stress.

The above method or use may comprise as abiotic stress drought,salinity, heat and/or chilling.

The cell wall invertase as referred to in the above mentioned method anduse may be any cell wall invertase without being restricted toChenopodium rubrum cell wall invertase or a homolog thereof or afunctional part thereof, with the function of hydrolysing sucrose intoglucose and fructose outside the cell which are then transported intocells and of conferring on a maize plant tolerance to abiotic stress.These functions also apply to the “part” and “homolog”, which areotherwise defined as outlined above. The definitions of the otherfeatures as comprised by the method or use such as tolerance, abioticstress, expression cassette, vector etc. are as comprised herein.

The invention is further explained in the following figures and exampleswhich are included for illustration purposes and are not intended tolimit the invention.

FIGURES

FIG. 1A-C: Vectors used for Cloning CrCIN: Three vectors were used forcloning CrCIN. A: The first vector was received from GeneArt(ThermoScientific) containing a synthesised codon-optimized CrCIN gene(SEQ ID NO: 3). B: This gene was excised using the restriction enzymesBamHI and HindIII and cloned into the shuttle vector pABM containing thecloning cassette (ubi promoter and NosT terminator). C: This entire genecassette was excised using the marked enzyme SfiI and cloned into thebinary vector pZFNmcherb for transformation into Agrobacterium andfinally maize.

FIG. 2: Expression levels of the homozygous T1 CrCIN plants: RT-qPCRdisplaying relative expression of selected CrCIN events to endogenouscontrol gene ZmEF1. Both non-transformed A188 and the transformationcontrol (A188 transformed with empty vector) showed no expression, whilethose A188-lines containing CrCIN as transgene showed CrCIN expression.

FIG. 3: T1 CrCIN plants winter 2015 (A) and T2 CrCIN plants winter 2016(B): Photographs of transgenic CrCIN plants, selected events of Event 1,Event 5, Event 8 and Event 9 (E1, E5, E8 and E9) lined up with 2controls, A188 WT (wildtype) and TC (transformation control) during 2growth periods at Week 9. Event 1 is absent from experiment in 2016. Allevents shown here displayed significant increases in yield (biomass).

FIG. 4: T1 CrCIN plant physiology measurements at week 8: Yieldcomparison of transgenic T1 CrCIN events E9, E5 and E8 plants comparedto A188 and transformation control plants (n=5) using the Iowa StateUniversity Vegetative Stage leaf counting method at 8 weeks aftersowing. Plants that were significantly different (student t-test)compared to A188 were marked with an asterisk while plants significantlydifferent to the transformation control were marked with a hash.

FIG. 5: T1 CrCIN plant physiology measurements at week 8: Plant heightcomparison of transgenic T1 CrCIN events E9, E5 and E8 plants comparedto A188 and transformation control plants (n=5) at 8 weeks after sowing.Plants that were significantly different (student t-test) compared toA188 were marked with an asterisk while plants significantly differentto the transformation control were marked with a hash.

FIG. 6: T2 plant physiology measurements at week 8: Yield comparison oftransgenic T2 CrCIN events E9, E5 and E8 plants (n=20) compared to A188and transformation control plants (n=40) using the Iowa State UniversityVegetative Stage leaf counting method at 8 weeks after sowing. Plantsthat were significantly different (student t-test) compared to A188 weremarked with an asterisk while plants significantly different to thetransformation control were marked with a hash.

FIG. 7: Experiment 1: CrCIN maize seedlings under simulated droughtstress: Graph displaying the percentage of leaves of 25% PEG6000 treatedversus untreated plants (n=10) that showed leaf rolling symptoms ofleaves. All plants were grown for 1 week in ¼ strength Hoagland Solutionand then treated for 1 day in added 25% PEG6000. Both control eventsshowed high levels of leaf rolling. Event 5 showed reduction in leafrolling symptoms. Event 8 and Event 9 showed a significant reduction inleaf rolling symptoms.

FIG. 8: Experiment 1: CrCIN maize seedlings under simulated droughtstress: Photo of Event 8 CrCIN seedlings in ¼ strength Hoagland for 1week after germination followed by 2 days treatment with 25% PEG6000.Here it can be seen that the leaves of Event 8 show less leaf rollingsymptoms than the WT.

FIG. 9: Experiment 1: CrCIN maize seedlings under simulated droughtstress: Photo of representative CrCIN plants after 2 days treatment with25% PEG6000 versus control grow in ¼ strength Hoagland solution. All theplants were grown first for 1 week in ¼ strength Hoagland aftergermination before being transferred to the 25% PEG6000.

FIG. 10: Experiment 2: CrCIN maize seedlings under simulated droughtstress: Graph displaying the percentage of leaves of 25% PEG6000 treatedversus untreated plants (n=10) that showed leaf rolling symptoms ofleaves. All plants were grown for 1 week in ¼ strength Hoagland Solutionafter germination and then treated for 1 day in added 25% PEG6000. Bothcontrol events showed high levels of leaf rolling, Event 5 and Event 9showed reduced levels of leaf rolling and Event 8 showed a significantreduction in leaf rolling symptoms.

FIG. 11: Experiment 2: CrCIN maize seedlings under simulated droughtstress: Photo of Event 8 CrCIN plants after 2 days treatment with 25%PEG6000. Here it can be seen that the leaves of Event 8 show less leafrolling symptoms than the WT. All the plants were grown first for 1 weekin ¼ strength Hoagland after germination before being transferred to the25% PEG6000.

FIG. 12: Experiment 2: CrCIN maize seedlings under simulated droughtstress: Photo of three representative CrCIN plants after 2 daystreatment with 25% PEG6000. The biggest difference is the development ofthe 3rd leaf in Event 8 and Event 9 plants versus the controls. All theplants were grown first for 1 week in ¼ strength Hoagland aftergermination before being transferred to the 25% PEG6000.

FIG. 13: Data from wheat CrCIN transgenic plants: A: Plasmid map ofwheat pABM-ubi-CrCIN (Apr_Ampicillin resistance) and B: Plasmid map ofwheat pLHAB-ubi-CrCIN (aadA: Spectinomycin resistance, ColE1 ori: originof replication for E. coli, pVS1 REP: origin of replication forAgrobacterium).

FIG. 14: Wheat: CrCIN T1 screening, CrCIN expression: Mean±SE. Fivebiological replicates were used. CrCIN expression was analyzed fromleaves of four week old plants grown in the greenhouse. Expression ofTaEF was used as internal control. All plants were fully randomized inthe greenhouse.

FIG. 15: CrCIN overexpression does not increase yield or yield-relatedparamenters in wheat in the greenhouse. (A) Ear length, (B) Grain numberper ear, (C) Grain weight per ear and (D) Grain weight was measured onthe 4 first matured tillers of greenhouse-grown plants. Shown areMeans±Standard error, N≥10 biological replicates. Statistical analysiswas done by Two-way Anova. Other growth parameters (e.g. plant height)also did not show any significant difference.

FIG. 16. CrCIN overexpression does not increase yield in the field.Numbers present yield in percentage of control plants (non-transgenicTAIFUN) at different locations. ANOVA analysis of single and multiplelocations did not reveal any significant difference between transgenicCrCIN lines and control.

FIG. 17. CrCIN overexpression in wheat (TAIFUN) does not lead to adetectable drought tolerance phenotype neither with respect to the leafdry mass (top) nor to the root dry mass (bottom). black column: controlwithout drought stress; white column: with drought stress simulated byapplication of 10% PEG; 1, 2, 3: transgenic CrCIN lines with CrCINoverexpression; 5 and 6: lines without CrCIN overexpression (control).

EXAMPLES Results with Transgenic Maize Plants

We first synthesized the Chenopodium rubrum cell wall invertase (CrCIN)gene and then transformed it into a shuttle vector cassette containingan ubiquitin promoter (containing an intron) from maize and a 35Sterminator sequence to induce constitutive overexpression of the gene ina corn plant (FIGS. 1A and B). This cassette was then transformed into abinary vector containing for instance a herbicide gene (e.g.: BASTAresistance, glyphosate resistance or ALS inhibitor resistance) andspectinomycin resistance gene for subsequent transformation intoAgrobacterium tumefaciens for Agrobacterium mediated planttransformation into maize (Zea mays) genotype A188 (FIG. 1C).

These subsequently transformed maize embryos were then selected byherbicide treatment and regenerated into plants for seed production inthe greenhouse. From this seed batch T1 homozygous plants were grown.The expression levels of the regenerated homozygous T1 CrCIN plants havebeen determined by means of RT-qPCR displaying relative expression ofselected CrCIN events to endogenous control gene ZmEF1 (FIG. 2). Bothnon-transformed A188 and the transformation control (A188 transformedwith empty vector) showed no expression, while those A188-linescontaining CrCIN as transgene showed CrCIN expression at differentlevels.

In addition, T1 homozygous plants were analyzed in the greenhouse forgeneral physiological changes using primarily the leaf stage protocolcomprising the counting of all leaves including the dead ones startingfrom the base of the plant to the first exposed leaf as per the IowaState University protocol—also known as Leaf Collar Method (Abendroth etal., 2011, Corn Growth and Development, Iowa State University, AvailableInventory: 9182).

The Leaf Collar Method determines leaf stage in corn by counting thenumber of leaves on a plant with visible leaf collars, beginning withthe lowermost, short, rounded-tip true leaf and ending with theuppermost leaf with a visible leaf collar. The leaf collar is thelight-colored collar-like “band” located at the base of an exposed leafblade, near the spot where the leaf blade comes in contact with the stemof the plant. Leaves within the whorl, not yet fully expanded and withno visible leaf collar are not included in this leaf staging method. Theexception to this statement may be that leaves with barely visible leafcollars can be counted when you are staging plants early in the day,recognizing that the leaf collar may become completely visible by theend of the day. Leaf stages are usually described as “V” stages, e.g.,V2=two leaves with visible leaf collars. The leaf collar method isgenerally the most widely used method by university and industryagronomists in the US. Mass accumulation in the CrCIN plants wasobserved to increase from the V8 stage of growth until reproductivestage compared to the control plants in all events that showedexpression (FIG. 3A). This was measured by counting the V stages of theplants where the transgenic plants had significantly more leaves thanthe control A188 plants (FIG. 4).

In one experiment, the plant height was also measured by bunching theleaves together and then pulling up and measuring plant height from thesoil/plant stem base to the top of the tallest leaf (FIG. 5).

T2 homozygous seeds collected from these plants were then grown a secondtime and the biomass phenotype was reconfirmed by determining V stagesat 8 weeks growth under greenhouse and field conditions (FIG. 6). Plantheight was not measured again in the T2 plants as this could be clearlyseen by eye (cf. FIG. 3B).

T2 seedlings were tested in a hydroponics experiment with 25% PEG6000 in0.25× strength Hoagland solution to simulate drought stress (osmoticstress). Under such drought stress corn seedlings usually develop severeleaf dehydration and leaf rolling symptoms. Thus, leaf rolling ingrasses like maize may be used as an estimate of obvious effects ofwater deficit (O'Toole, John C., and Rolando T. Cruz. “Response of leafwater potential, stomatal resistance, and leaf rolling to water stress.”Plant physiology 65.3 (1980): 428-432.). Investigating the levels ofleaf rolling the seedlings with CrCIN Events E5, E8 and E9 showedenhanced tolerance to PEG6000 application compared to control A188plants and transformation control in replicated experiments (experiment1: FIGS. 7-9; experiment 2: FIGS. 10-12). of T2 seedlings (FIGS. 7-12).In these experiments there seems to be a dosage effect with the highestexpressing events showing the strongest phenotype. As can be seen fromthe experiments, all maize plants into which the CrCIN nucleic acid hasbeen introduced and which express CrCIN produce an increased yield undernormal and drought conditions and have the drought tolerant phenotype.

Negative Results with Transgenic Wheat Plants

CrCIN was overexpressed in wheat using an ubiquitin promotor(pABM-ubi-CrCIN and pLHAB-ubi-CrCIN; FIGS. 13A and B). Homozygous T1plants were screened in the greenhouse. CrCIN expression was analyzedfrom leaves of four week old plants grown in the greenhouse. Expressionof TaEF was used as internal control. All plants were fully randomizedin the greenhouse. The non-transgenic control (TAIFUN transformed withempty vector) showed no expression, while those TAIFUN-lines containingCrCIN as transgene showed CrCIN expression at different levels. However,in contrast to the results observed in maize CrCIN overexpression inwheat surprisingly does not increase yield or yield-related parametersin the greenhouse. Even though different types of yield measurementshave been executed, e.g., measuring plant height (data not shown), earlengths (FIG. 15A), counting grain number per ear (FIG. 15B), measuringgrain weight per ear (FIG. 15C) and grain weight measured on the 4 firstmatured tillers of greenhouse-grown plants, no significant differencehave been determined. Measurements have been repeated with T2 and T3lines in greenhouse and field. Field trials were done at 5 differentlocations with randomized complete block design (RCBD) in 4 replicates.even these trials revealed no significant difference in yield whencompared to non-transgenic background TAIFUN (FIG. 16).

Furthermore, the CrCIN overexpression in wheat does not show asignificant effect on potential drought tolerance in wheat. There is nodetectable difference in leaf dry mass or root dry mass between CrCINoverexpression lines and control lines without CrCIN overexpression inresponse to drought stress by PEG application (FIG. 17).

The invention claimed is:
 1. A transgenic maize plant comprising astransgene stably integrated into the genome of the maize plant: i) anexpression cassette comprising a nucleic acid capable of expressing aChenopodium rubrum cell wall invertase according to SEQ ID NO: 3,wherein the expression of the nucleic acid is controlled by an ubiquitinpromoter comprising an intron and a 35S terminator sequence inducingconstitutive overexpression of more than 10 fold relative to endogenouscontrol gene ZmEF1, and wherein as a result of the expression of thecell wall invertase the transgenic maize plant exhibits an enhancedtolerance to drought and an increased biomass yield, optionally ascompared to a reference.
 2. A plant cell, a tissue, a harvestable partor a seed of the transgenic maize plant of claim 1, wherein the plantcell, the tissue, the part or the seed comprises the transgene.
 3. Amethod of producing the transgenic maize plant of claim 1, comprisingthe following steps: introducing into at least a cell of a maize plantthe expression cassette according to claim 1, or a vector comprising theexpression cassette, and regenerating the transgenic maize plant fromthe at least a cell.
 4. A method of enhancing the drought tolerance of amaize plant and of increasing biomass yield potential of a maize plant,comprising the following steps: introducing into at least a cell of amaize plant expression cassette according to claim 1, or a vectorcomprising the expression cassette, and causing constitutiveoverexpression of more than 10 fold relative to endogenous control geneZmEF1.
 5. A vector comprising the expression cassette according toclaim
 1. 6. A method for production of ethanol or methane comprising thefollowing steps: cutting the transgenic maize plant according to claim1, optionally treating the cut maize plant with an ensilage agent,optionally storing the cut maize plant optionally treated with anensilage agent, and producing ethanol or methane from the cut maizeplant by anaerobic digestion.
 7. A method for production of ethanol ormethane comprising the following steps: cutting the harvestable partaccording to claim 2, optionally treating the cut harvestable part withan ensilage agent, optionally storing the cut harvestable partoptionally treated with an ensilage agent, and producing ethanol ormethane from the cut harvestable part by anaerobic digestion.